Methods of synthesizing nicotinamide riboside

ABSTRACT

Provided herein are efficient and scalable methods for the synthesis of nicotinamide riboside from riboside triacetate. Also provided are compositions comprising nicotinamide riboside, and therapeutic methods employing nicotinamide riboside.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/527,382, filed Jun. 30, 2017, whichapplication is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Nicotinamide riboside is a pyridine-nucleoside form of niacin (i.e.,vitamin B₃) that serves as a precursor to nicotinamide adeninedinucleotide (NAD⁺). NAD⁺ promotes cellular metabolism, mitochondrialfunction, and energy production. Currently, nicotinamide riboside ismade through synthetic methods or fermentation processes. Because of itssignificant potential to confer health benefits when used as a dietarysupplement, there exists a need to develop highly efficient and scalableprocesses for the manufacture of nicotinamide riboside.

SUMMARY OF THE INVENTION

In some aspects, the present invention provides a method of preparingcompound 3, comprising the steps of:

-   (a) combining compound 1 and a chlorinating agent, thereby producing    a first reaction mixture comprising compound 2, wherein:    -   compound 1 is represented by

-   -   compound 2 is represented by

and

-   -   the chlorinating agent is gaseous HCl or HCl in dioxane; and

-   (b) combining compound 2 with nicotinamide, thereby producing a    second reaction mixture comprising compound 3.

In further aspects, the present invention provides a method of preparingcompound 4, comprising the steps of:

-   (c) combining compound 3 with an ammonia reagent, thereby producing    a first reaction mixture comprising compound 4, wherein:    compound 3 is represented by

andthe ammonia reagent is methanolic ammonia or diethylamine.

In further embodiments, the invention provides a compound having thestructure of Compound 4, made according to method described herein.

In certain aspects, the present invention provides a method of improvingcellular health in a subject, comprising administering to the subject acompound having the structure of Compound 4, made according to methoddescribed herein.

In certain aspects, the present invention provides a method of improvingsleep quality, stimulating or increasing REM sleep, or treating orpreventing insomnia, desynchronosis, or a circadian rhythm sleepdisorder in a subject, comprising administering to the subject acompound having the structure of Compound 4, made according to methoddescribed herein.

In certain aspects, the present invention provides a method of treatingor preventing a motor neuron disease or ALS, or slowing or reversing theprogression of motor neuron degeneration in a subject, comprisingadministering to the subject a compound having the structure of Compound4, made according to method described herein.

In certain aspects, the present invention provides a method of improvingfertility, treating or preventing infertility, inducing ovulation,increasing sperm count, or increasing lactation, comprisingadministering to the subject a compound having the structure of Compound4, made according to method described herein.

In certain aspects, the present invention provides a method of treatingor preventing kidney damage, acute kidney injury, or kidney disease, orincreasing blood flow to the kidneys, comprising administering to thesubject a compound having the structure of Compound 4, made according tomethod described herein.

In certain aspects, the present invention provides a method of treatingor preventing liver damage or fatty liver, or decreasing the serum levelof alanine transaminase (ALT) or aspartate transaminase (AST) in asubject, comprising administering to the subject a compound having thestructure of Compound 4, made according to method described herein.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbyl-C(O)—, preferably alkyl-C(O)—.

The term “acetyl” refers to a specific acyl group having the formulaH₃C—C(O)—. Acetyl may be abbreviated as “Ac-.”

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10, or 1 to 6, unless otherwisedefined. Examples of straight chained and branched alkyl groups includemethyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl,pentyl, hexyl, pentyl and octyl. A C₁-C₆ straight chained or branchedalkyl group is also referred to as a “lower alkyl” group.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas alkyl is meant to include groups that contain from x to y carbons inthe chain. For example, the term “C_(x-y)alkyl” refers to substituted orunsubstituted saturated hydrocarbon groups, including straight-chainalkyl and branched-chain alkyl groups that contain from x to y carbonsin the chain, including haloalkyl groups such as trifluoromethyl and2,2,2-tirfluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

A “trialkylamine” means a compound having the structure N(R¹)(R²)(R³),wherein R¹, R², and R³ are each an alkyl group, and can be identical ordifferent. Exemplary trialkylamine compounds include trimethylamine,trimethylamine, triisopropylamine, tributylamine, andisopropyldimethylamine.

As used herein, the term “chlorinating agent” means a reagent thatprovides a source of chlorine anions (i.e., Cl⁻). Exemplary chlorinatingagents include sources of HCl, such as HCl gas or HCl in a solvent suchas ethanol or dioxane. For example, a chlorinating agent used in themethods of the invention is preferably HCl in dioxane. An alternativechlorinating agent that is preferably used in some embodiments of themethods of the invention is gaseous HCl (i.e., HCl (gas)).

Nicotinamide is a molecule having the following structure:

As used herein, the term “ammonia reagent” means a reagent that providesa source of ammonia (i.e., NH₃), or an alkyl amine (such asmethylamine), or a dialkylamine (such as diethylamine), or analkylenediamine (such as ethylenediamine). Ammonia may be supplied as agas (i.e., NH₃ (g)) or in a solution of an organic solvent, such asmethanol, ethanol, isopropanol, dioxane, or tetrahydrofuran (THF). Insome embodiments, the term “ammonia reagent” also includes ammoniasupplied in aqueous solution.

In describing a process of filtration, the liquid material that passesthrough the filter is referred to herein as the “filtrate”. The solidmaterial that collects (i.e., is filtered from the filtrate), isreferred to herein as the “retentate.”

As used herein, the term “administering” means providing apharmaceutical agent or composition to a subject, and includes, but isnot limited to, administering by a medical professional andself-administering. Administration of a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered, intravenously, arterially, intradermally,intramuscularly, intraperitoneally, subcutaneously, ocularly,sublingually, orally (by ingestion), intranasally (by inhalation),intraspinally, intracerebrally, and transdermally (by absorption, e.g.,through a skin duct). A compound or agent can also appropriately beintroduced by rechargeable or biodegradable polymeric devices or otherdevices, e.g., patches and pumps, or formulations, which provide for theextended, slow or controlled release of the compound or agent.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

Appropriate methods of administering a substance, a compound or an agentto a subject will also depend, for example, on the age and/or thephysical condition of the subject and the chemical and biologicalproperties of the compound or agent (e.g., solubility, digestibility,bioavailability, stability and toxicity). In some embodiments, acompound or an agent is administered orally, e.g., to a subject byingestion. In some embodiments, the orally administered compound oragent is in an extended release or slow release formulation, oradministered using a device for such slow or extended release.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial.

As used herein, the term “subject” means a human or non-human animalselected for treatment or therapy.

The phrases “therapeutically-effective amount” and “effective amount” asused herein means the amount of an agent which is effective forproducing the desired therapeutic effect in at least a sub-population ofcells in a subject at a reasonable benefit/risk ratio applicable to anymedical treatment.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that at least one symptom ofthe disease is decreased or prevented from worsening.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, when administered to a statistical sampleprior to the onset of the disorder or condition, reduces the occurrenceof the disorder or condition in the treated sample relative to anuntreated control sample, or delays the onset or reduces the severity ofone or more symptoms of the disorder or condition relative to theuntreated control sample.

II. Methods of the Invention

This invention is based on the discovery of an efficient, scalablesynthesis of nicotinamide riboside.

In certain aspects, the present invention provides a method forpreparing compound 3,

comprising the steps of:

-   (a) combining compound 1 and a chlorinating agent, thereby producing    a first reaction mixture comprising compound 2, wherein:    -   compound 1 is represented by

-   -   compound 2 is represented by

and

-   -   the chlorinating agent is gaseous HCl or HCl in dioxane; and

-   (b) combining compound 2 with nicotinamide, thereby producing a    second reaction mixture comprising compound 3.

In certain embodiments, the chlorinating agent is gaseous HCl (i.e., HCl(g)). Alternatively, in some embodiments, the chlorinating agent is HClin dioxane.

In certain embodiments, step (a) occurs in a first solvent. The firstsolvent may be combined with compound 1 (i.e., a solution of compound 1in the first solvent may be produced) prior to combination of compound 1with the chlorinating agent. Alternatively, the first solvent, compound1, and the chlorinating agent may be combined together simultaneously.In another alternative, the first solvent and the chlorinating agent maybe combined first, followed by combination of that mixture with compound1.

In some embodiments, the first solvent is a component of thechlorinating agent. For example, in certain embodiments the chlorinatingagent is HCl in dioxane. In certain such embodiments, dioxane is thefirst solvent.

In certain embodiments, the chlorinating agent is HCl in dioxane and thefirst solvent is dioxane. In alternative embodiments, the chlorinatingagent is HCl in dioxane and the first solvent is dichloromethane (DCM).

Alternatively, in methods in which the chlorinating agent is gaseousHCl, the first solvent may be acetonitrile.

In certain embodiments, step (a) further comprises acetyl chloride(AcCl).

Step (a) may be monitored by any reaction monitoring mechanism employedby persons of ordinary skill in the art, including thin layerchromatography, nuclear magnetic resonance spectroscopy, massspectroscopy, and high performance liquid chromatography.

Upon completion of step (a), the reaction mixture may be concentrated toremove or reduce the amounts of HCl and excess solvent present in thereaction mixture. In some embodiments, such a concentration step affordsa concentrated crude material comprising compound 2.

Thus, in certain embodiments, the method further comprises step (a′):

(a′) concentrating the first reaction mixture under reduced atmosphericpressure; wherein step (a′) occurs after step (a) and before step (b).

Upon completion of steps (a) and (a′), a concentrated crude materialcomprising compound 2 is afforded.

In certain embodiments, the methods of the invention further comprisecombining compound 2 and a second solvent. For example, the concentratedcrude material comprising compound 2 yielded from steps (a) and (a′) maybe combined with a second solvent.

In certain embodiments, the second solvent is acetonitrile.

In certain embodiments, step (b) occurs in a second solvent. The secondsolvent may be a polar aprotic solvent. For example, the second solventmay be acetonitrile.

In certain embodiments, the method further comprises step (b′):

(b′) combining the second reaction mixture with a trialkylamine;wherein step (b′) occurs after step (b).

In certain such embodiments, the trialkylamine is tributylamine.

In certain embodiments, following step (b′), the resultant reactionmixture comprising compound 3 is filtered, and the solids (i.e.,retentate) are washed. The retentate comprises compound 3. The retentatemay be washed with a cold solvent, such as cold acetonitrile or coldmethanol.

In certain embodiments, the method further comprises step (c):

(c) combining compound 3 with an ammonia reagent, thereby producing athird reaction mixture comprising compound 4, wherein:compound 4 is represented by

andthe ammonia reagent is methanolic ammonia or diethylamine.

In certain embodiments, the ammonia reagent is diethylamine.

In certain embodiments, the ammonia reagent is methanolic ammonia.

In certain embodiments, the methanolic ammonia is 1M, 2M, 3M, 4M, 5M,6M, 7M, 8M, 9M, or 10M methanolic ammonia. Preferably, the methanoicammonia is 7M methanolic ammonia.

In certain embodiments, step (c) occurs in a third solvent. The thirdsolvent may be a polar protic solvent, such as an alcohol. The thirdsolvent may, for example, be methanol, ethanol, isopropanol, or butanol.In certain preferred embodiments, the third solvent is methanol.

In certain embodiments, step (c) takes place at a temperature below roomtemperature.

In further embodiments wherein the ammonia reagent is diethylamine, themethod of the invention further comprises step (c′), which occurs afterstep (c):

-   (c′) adding cold aqueous HCl to the third reaction mixture, thereby    producing a fourth mixture comprising compound 4.

In further embodiments wherein the ammonia reagent is methanolicammonia, the method of the invention further comprises step (c′), whichoccurs after step (c):

-   (c′) concentrating the third reaction mixture under reduced    atmospheric pressure, thereby producing a fourth mixture comprising    compound 4.

In certain embodiments, the method of the invention further comprisesstep (c″), which occurs after step (c′):

-   (c″) filtering the fourth mixture.

In certain such embodiments, the method further comprises rinsing theresultant retentate (i.e., the solids left behind after filtration) withcold methanol or ethanol.

In other certain such embodiments, the method further comprises rinsingthe resultant retentate (i.e., the solids left behind after filtration)with cold acetone.

In yet other certain such embodiments, the method further comprisesrinsing the resultant retentate (i.e., the solids left behind afterfiltration) with cold methyl tert butyl ether (MTBE).

In certain embodiments, the reaction between the ammonia reagent andcompound 3 may be monitored by any reaction monitoring mechanismemployed by persons of ordinary skill in the art. Upon completion orsignificant progression of this reaction (i.e., upon formation ofcompound 4), the reaction mixture may be distilled, for example toremove the ammonia reagent and the third solvent. In certain suchembodiments, compound 4 remains in the distillation pot. In further suchembodiments, the remaining contents of the distillation may then byfiltered and washed. In certain embodiments, the washing liquid is coldacetone and/or cold methanol.

In further embodiments, the invention provides a method of preparingcompound 4,

comprising the steps of:(c) combining compound 3 with an ammonia reagent, thereby producing afirst reaction mixture comprising compound 4, wherein:compound 3 is represented by

andthe ammonia reagent is methanolic ammonia or diethylamine.

In certain embodiments, the ammonia reagent is diethylamine.

In certain embodiments, the ammonia reagent is methanolic ammonia.

In certain embodiments, the methanolic ammonia is 1M, 2M, 3M, 4M, 5M,6M, 7M, 8M, 9M, or 10M methanolic ammonia. Preferably, the methanoicammonia is 7M methanolic ammonia.

In certain embodiments, step (c) occurs in a third solvent. The thirdsolvent may be a polar protic solvent, such as an alcohol. The thirdsolvent may, for example, be methanol, ethanol, isopropanol, or butanol.In certain preferred embodiments, the third solvent is methanol.

In certain embodiments, step (c) takes place at a temperature below roomtemperature.

In further embodiments wherein the ammonia reagent is diethylamine, themethod of the invention further comprises step (c′), which occurs afterstep (c):

-   (c′) adding cold aqueous HCl to the third reaction mixture, thereby    producing a fourth mixture comprising compound 4.

In further embodiments wherein the ammonia reagent is methanolicammonia, the method of the invention further comprises step (c′), whichoccurs after step (c):

-   (c′) concentrating the third reaction mixture under reduced    atmospheric pressure, thereby producing a fourth mixture comprising    compound 4.

In certain embodiments, the method of the invention further comprisesstep (c″), which occurs after step (c′):

-   (c″) filtering the fourth mixture.

In certain such embodiments, the method further comprises rinsing theresultant retentate (i.e., the solids left behind after filtration) withcold methanol or ethanol.

In other certain such embodiments, the method further comprises rinsingthe resultant retentate (i.e., the solids left behind after filtration)with cold acetone.

In yet other certain such embodiments, the method further comprisesrinsing the resultant retentate (i.e., the solids left behind afterfiltration) with cold methyl tert butyl ether (MTBE).

In certain embodiments, the method further comprises:

-   (a) combining compound 1 and a chlorinating agent, thereby producing    a first reaction mixture comprising compound 2, wherein:    -   compound 1 is represented by

-   -   compound 2 is represented by

and

-   -   the chlorinating agent is gaseous HCl or HCl in dioxane; and

-   (b) combining compound 2 with nicotinamide, thereby producing a    second reaction mixture comprising compound 3.

In certain embodiments, the chlorinating agent is gaseous HCl (i.e., HCl(g)). Alternatively, in some embodiments, the chlorinating agent is HClin dioxane.

In certain embodiments, step (a) occurs in a first solvent. The firstsolvent may be combined with compound 1 (i.e., a solution of compound 1in the first solvent may be produced) prior to combination of compound 1with the chlorinating agent. Alternatively, the first solvent, compound1, and the chlorinating agent may be combined together simultaneously.In another alternative, the first solvent and the chlorinating agent maybe combined first, followed by combination of that mixture with compound1.

In some embodiments, the first solvent is a component of thechlorinating agent. For example, in certain embodiments the chlorinatingagent is HCl in dioxane. In certain such embodiments, dioxane is thefirst solvent.

In certain embodiments, the chlorinating agent is HCl in dioxane and thefirst solvent is dioxane. In alternative embodiments, the chlorinatingagent is HCl in dioxane and the first solvent is dichloromethane (DCM).

Alternatively, in methods in which the chlorinating agent is gaseousHCl, the first solvent may be acetonitrile.

In certain embodiments, step (a) further comprises acetyl chloride(AcCl).

Step (a) may be monitored by any reaction monitoring mechanism employedby persons of ordinary skill in the art, including thin layerchromatography, nuclear magnetic resonance spectroscopy, massspectroscopy, and high pressure liquid chromatography.

Upon completion of step (a), the reaction mixture may be concentrated toremove or reduce the amounts of HCl and excess solvent present in thereaction mixture. In some embodiments, such a concentration step affordsa concentrated crude material comprising compound 2.

Thus, in certain embodiments, the method further comprises step (a′):

(a′) concentrating the first reaction mixture under reduced atmosphericpressure; wherein step (a′) occurs after step (a) and before step (b).

Upon completion of steps (a) and (a′), a concentrated crude materialcomprising compound 2 is afforded.

In certain embodiments, the methods of the invention further comprisecombining compound 2 and a second solvent. For example, the concentratedcrude material comprising compound 2 yielded from steps (a) and (a′) maybe combined with a second solvent.

In certain embodiments, the second solvent is acetonitrile.

In certain embodiments, step (b) occurs in a second solvent. The secondsolvent may be a polar aprotic solvent. For example, the second solventmay be acetonitrile.

In certain embodiments, the method further comprises step (b′):

(b′) combining the second reaction mixture with a trialkylamine;wherein step (b′) occurs after step (b).

In certain such embodiments, the trialkylamine is tributylamine.

In certain embodiments, following step (b′), the resultant reactionmixture comprising compound 3 is filtered, and the solids (i.e.,retentate) are washed. The retentate comprises compound 3. The retentatemay be washed with a cold solvent, such as cold acetonitrile or coldmethanol.

A “polar protic solvent” as used herein is a solvent having a dipolemoment of about 1.4 to 4.0 D, and comprising a chemical moiety thatparticipates in hydrogen bonding, such as an O—H bond or an N—H bond.Exemplary polar protic solvents include methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, ammonia, water, and acetic acid.

A “polar aprotic solvent” as used herein means a solvent having a dipolemoment of about 1.4 to 4.0 D that lacks a hydrogen bonding group such asO—H or N—H. Exemplary polar aprotic solvents include acetone,N,N-dimethylformamide, acetonitrile, ethyl acetate, dichloromethane,tetrahydrofuran, and dimethyl sulfoxide.

A “non-polar solvent” as used herein means a solvent having a lowdielectric constant (<5) and low dipole moment of about 0.0 to about1.2. Exemplary nonpolar solvents include pentane, hexane, cyclohexane,benzene, toluene, chloroform, and diethyl ether.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Examples of alcohols include, without limitation, methanol, ethanol,2-butoxyethanol, propanol, allyl alcohol, methallyl alcohol, prenol,isopropanol, 2,2-dimethylpropan-1-ol, 2-methyl-2-phenylpropan-1-ol,butanol, isobutanol, sec-butanol, tert-butanol, 2-buten-1-ol, pentanol,2-cyclopenten-1-ol, 4-cyclopenten-1-ol, cyclopentanol,3-cyclopenten-1-ol, hexanol, cyclohexanol, 3-cyclohexen-1-ol, phenol,1-naphthol, 2-naphthol, benzyl alcohol, menthol, 1,2-ethanediol,9-fluorenylmethanol, resorcinol, meta-cresol, cinnamyl alcohol, andgeraniol.

III. Compositions of the Invention

In certain embodiments, the invention provides Compound 4, madeaccording to any of the methods of preparation described herein.

In certain embodiments, compound 4 is produced in a yield of about 30%,about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about44%, about 46%, about 48%, about 50% or higher from compound 1.

In certain embodiments, the purity of the compound is 95%, 96%, 97%,98%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or higher.

As described in detail below, the pharmaceutical compositions describedherein may be specially formulated for administration in solid or liquidform, including those adapted for the following: (1) oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; (2) parenteral administration, for example,by subcutaneous, intramuscular, intravenous or epidural injection as,for example, a sterile solution or suspension, or sustained-releaseformulation; or (3) sublingually. In some embodiments, the compositioncomprises additional agents. For example, the composition may comprise anutritional agent, such as an antioxidant. Examples ofpharmaceutically-acceptable antioxidants include: (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The formulations of the compounds described herein may be presented inunit dosage form and may be prepared by any methods well known in theart of pharmacy. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will varydepending upon the host being treated and the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the agent which produces a therapeutic effect.

In certain embodiments, a formulation described herein comprises anexcipient, including, but not limited to, cyclodextrins, liposomes,micelle forming agents, e.g., bile acids, and polymeric carriers, e.g.,polyesters and polyanhydrides; and an agent of the invention. In someembodiments, an aforementioned formulation renders orally bioavailablean agent of the invention. Methods of preparing these formulations orcompositions may include the step of bringing into association acompound of the invention with the carrier and, optionally, one or moreaccessory ingredients.

Liquid dosage forms for oral administration of the formulations providedherein include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations provided herein suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of theinvention as an active ingredient. A compound of the invention may alsobe administered as a bolus, electuary, or paste.

In solid dosage forms of the invention for oral administration (e.g.,capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; (8) absorbents, such as kaolinand bentonite clay; (9) lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions described herein, such as dragees, capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. Compositions described herein may also beformulated for rapid release, e.g., freeze-dried. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Pharmaceutical compositions provided herein suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

III. Methods of Treatment

In certain embodiments, the present invention provides a method ofimproving cellular health in a subject, comprising administering to thesubject a compound made by the synthetic methods disclosed herein.

In further embodiments, the present invention provides methods for:

improving sleep quality;

stimulating or increasing REM sleep;

treating or preventing insomnia;

treating or preventing desynchronosis; or treating or preventing acircadian rhythm sleep disorder in a subject, comprising administeringto the subject Compound 4 prepared by the synthetic methods disclosedherein.

In certain embodiments, such a method of treatment further comprisesadministering pterostilbene.

In some embodiments, the methods treat or prevent a circadian rhythmsleep disorder. The circadian rhythm sleep disorder can be extrinsic(e.g., shift work sleep disorder, desynchornosis) or intrinsic (e.g.,advanced sleep phase disorder (ASPD), delayed sleep phase disorder(DSPD), irregular sleep-wake rhythm, and/or non-24-hour sleep-wakedisorder (i.e., hypernychthemeral syndrome)).

The subject may be male or female. In some embodiments, the subject isan adult (i.e., 18 years of age or older). The subject may be pediatric(i.e., less than 18 years of age). In some embodiments, the subject is amammal, preferably, a human.

In certain aspects, the methods and compositions provided herein relateto improving sleep health and the quality of sleep in a subject in asubject by administering to the subject (e.g., orally administering tothe subject) a composition comprising nicotinamide riboside (compound 4)and/or pterostilbene. Sleep quality may refer to the “restfulness” ofsleep (i.e., how rested an individual feels during waking hours). Sleepquality may refer to the quantity of sleep. Good sleep quality isassociated with a wide range of positive outcomes such as better health,less daytime sleepiness, greater well-being and better psychologicalfunctioning. In some embodiments, a subject has a score of 1, 2, or 3 onthe Pittsburgh Sleep Quality Index (PSQI). Further details on the PSQImay be found at Buysse, D. J., Reynolds, C. F., Monk, T. H., Berman, S.R., & Kupfer, D. J. (1989). The Pittsburgh Sleep Quality Index (PSQI): Anew instrument for psychiatric research and practice. PsychiatryResearch, 28(2), 193-213, incorporated herein by reference in itsentirety. In some embodiments, the subject has trouble falling asleep.In some embodiments, the subject has trouble staying asleep. In someembodiments, the subject wakes in the morning at an hour that woulddisrupt a normal sleep cycle or otherwise affect sleep quality.

In some aspects, provided herein are methods of stimulating REM sleep inneed thereof, comprising administering to the subject a compositioncomprising nicotinamide riboside (compound 4) and/or pterostilbene.Rapid eye movement sleep (REM sleep) is a unique phase of sleepcharacterized by rapid movement of the eyes, low muscle tone throughoutthe body, and the propensity of the sleeper to dream vividly. In someembodiments, the compositions and methods disclosed herein increase thetotal amount of time a subject is in REM sleep per sleep session (e.g.,the total amount of time in REM per night). In some embodiments, thecompositions and methods disclosed herein increase the amount of time asubject is in REM sleep per sleep cycle. Sleep progresses in a series offour or five more or less regular sleep cycles of non-REM and REM sleepthroughout the night. The first sleep cycle is typically around 90minutes in length, with the succeeding cycles averaging around 100-120minutes, although some individuals may have longer or shorter averagecycles. Each cycle follows the stages of non-REM sleep (stage 1-stage2-stage 3) and then, after a period in deep stage 3 slow-wave sleep,back through the stages (stage 3-stage 2-stage 1). Then, instead ofwaking, the sleeper may enter a short period of REM sleep, before goingback through non-REM stages in a new cycle. As the night progresses, thetime spent in deep stage 3 sleep decreases and the time spent in REMsleep increases, so that there is a greater proportion of stage 3 sleepearlier in the night, and a greater proportion of REM sleep later in thenight, particularly during the final two sleep cycles. As used herein,stimulating REM or increasing REM may refer to increasing the time asubject stays in REM sleep per sleep cycle or the total amount of time asubject is sleeping per day.

In certain aspects, the methods and compositions provided herein relateto the treatment and/or prevention of sleep disorders in a subject byadministering to the subject (e.g., orally administering to the subject)a composition comprising nicotinamide riboside (compound 4) and/orpterostilbene.

In some embodiments, the subject has insomnia. Insomnia is a sleepdisorder that is characterized by the inability to sleep. For example, asubject with insomnia may have trouble falling asleep, staying asleep,or wake up too early and not be able to get back to sleep. As usedherein, insomnia may refer to short-term (acute) insomnia (i.e., theinability to sleep that lasts for days to weeks) or long-term (chronic)insomnia (i.e., the inability to sleep for one month or more). In someembodiments, the insomnia is transient insomnia. Insomnia may be theresult of stress, a traumatic event, nasal/sinus allergies,gastrointestinal problems, brain lesions and tumors, stroke, chronicpain, chronic fatigue syndrome, congestive heart failure, angina,acid-reflux disease (GERD), chronic obstructive pulmonary disease,asthma, endocrine disorders such as hyperthyroidism, arthritis,neurological conditions such as Parkinson's or Alzheimer's disease, lowback pain, or genetics. In some embodiments, the subject has restlessleg syndrome. In some embodiments, the subject has sleep apnea. In someembodiments, the subject has a psychological condition that interfereswith sleep, such as anxiety, depression, bipolar disorder,schizophrenia, posttraumatic stress disorder (PTSD), and/or attentiondeficit hyperactivity disorder (ADHD). In some embodiments, insomnia isa side effect of medication. Examples of medications that may causeinsomnia include, but are not limited to, corticosteroids, alphablockers, beta blockers, SSRI antidepressants, ACE inhibitors,cholinesterase inhibitors, second generation (non-sedating) HI agonists,or glucosamine/chondroitin.

Provided herein are methods and compositions useful in regulating asubject's circadian rhythm. Circadian rhythms regulate the timing ofperiods of sleepiness and wakefulness throughout the day. Circadianrhythms are endogenously generated, although they can be modulated byexternal cues such as sunlight and temperature. Circadian rhythms areimportant in determining the sleeping and feeding patterns of allanimals, including human beings. There are clear patterns of brain waveactivity, hormone production, cell regeneration and other biologicalactivities linked to this daily cycle, and an irregular circadian rhythmmay lead to a disturbance any of the previously mentioned processes. Insome embodiments, the subject has a circadian rhythm sleep disorder. Thecircadian rhythm sleep disorder may be extrinsic (e.g., the result ofenvironmental influences or circumstances) or intrinsic (e.g., theresult of genetics or not the result of circumstances). An example of anextrinsic circadian sleep disorder includes shift work sleep disorder,which often affects individuals who work nights or in rotating shifts.Intrinsic sleep disorders include advanced sleep phase disorder (ASPD),delayed sleep phase disorder (DSPD), irregular sleep-wake rhythm, and/ornon-24-hour sleep-wake disorder (i.e., hypernychthemeral syndrome)).ASPD is characterized by difficulty staying awake in the evening anddifficulty staying asleep in the morning. DSPD is characterized by amuch later than normal timing of sleep onset and offset and a period ofpeak alertness in the middle of the night. Individuals with irregularsleep-wake rhythm suffer from sleeping at very irregular times, andusually more than twice per day (waking frequently during the night andtaking naps during the day), but often sleep a normal period of totaltime per day typical for the person's age. Non-24-hour sleep-wakedisorder, or hypernychthemeral syndrome, is a sleep disorder wherein theaffected individual's sleep occurs later and later each day, with theperiod of peak alertness also continuously moving around the clock fromday to day.

In some aspects, the compositions and methods provided herein are usefulin treating desynchronosis (i.e., jet lag). Jet lag is a temporary sleepdisorder caused by crossing time zones (e.g., during an airplaneflight), and is often the result of disruption to the circadian rhythmsof the body. Jet lag may occur any time the body's internal clock is outof sync with cues from a new time zone. Cues can include light exposureand eating times. General symptoms include fatigue and disorientation,interrupted sleep, confusion, mood changes, and pain in limbs.

Actual dosage levels and administration regimen of the compositionsdisclosed herein may be varied so as to obtain an amount of nicotinamideriboside and/or pterostilbene that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. In some embodiments,the subject continuously self-administers the compounds disclosedherein. In other embodiments, the subject may take a compound disclosedherein as needed.

In some embodiments, administration of the composition comprisesadministration of the composition in one or more dose(s). In someembodiments, administration of the composition comprises administrationof the composition in one or more, five or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, one hundred or more,or one thousand or more dose(s). In some embodiments, the dose comprisesat least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, atleast 125 mg, at least 150 mg, at least 200 mg, at least 225 mg, atleast 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, atleast 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, atleast 450 mg, at least 475 mg, at least 500 mg, at least 550 mg, atleast 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, atleast 800 mg, or at least 850 mg nicotinamide riboside (compound 4). Insome embodiments, the dose comprises at least 5 mg, at least 10 mg, atleast 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, atleast 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 110 mg, atleast 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, atleast 160 mg, least 170 mg, at least 180 mg, at least 190 mg, at least200 mg, or at least 250 mg of pterostilbene.

The compositions disclosed herein may be administered over any period oftime effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. The period of time may be at least 1 day, atleast 10 days, at least 20 days, at least 30, days, at least 60 days, atleast three months, at least six months, at least a year, at least threeyears, at least five years, or at least ten years. The dose may beadministered when needed, sporadically, or at regular intervals. Forexample, the dose may be administered monthly, weekly, biweekly,triweekly, once a day, or twice a day.

In further embodiments, the present invention provides methods of:

treating or preventing a motor neuron disease;

treating or preventing ALS; or

slowing or reversing the progression of motor neuron degeneration in asubject, comprising administering to the subject Compound 4 prepared bythe synthetic methods disclosed herein.

In certain embodiments, such a method of treatment further comprisesadministering pterostilbene.

In certain embodiments, the motor neuron disease is ALS, hereditaryspastic paraplegia (HSP), primary lateral sclerosis (PLS), progressivemuscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbarpalsy, or a spinal muscular atrophy.

In some embodiments, the subject may have or be predisposed to a motorneuron disease (e.g., amyotrophic lateral sclerosis (ALS), such asmedulla ALS or brainstem ALS). A motor neuron disease or disorder may beany disease or disorder that affects the function or structure of motorneuron. As used herein, a motor neuron diseases include progressivediseases that result in loss of function of motor neurons, or nerves, inthe brain and spinal cord. Examples of motor neuron diseases includeamyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia(HSP), primary lateral sclerosis (PLS), progressive muscular atrophy(PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, or a spinalmuscular atrophy. A motor neuron disease may affect the upper motorneurons or the lower motor neurons.

Actual dosage levels and administration regimen of the compositionsdisclosed herein may be varied so as to obtain an amount of nicotinamideriboside (compound 4) and/or pterostilbene that is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

In some embodiments, administration of the composition comprisesadministration of the composition in one or more dose(s). In someembodiments, administration of the composition comprises administrationof the composition in one or more, five or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, one hundred or more,or one thousand or more dose(s). In some embodiments, the dose comprisesat least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, atleast 500 mg, at least 600 mg, at least 700 mg, at least 800 mg, atleast 900 mg, at least 1000 mg, at least 1100 mg, at least 1200 mg, atleast 1300 mg, at least 1400 mg, at least 1500 mg, at least 1600 mg, atleast 1700 mg, at least 1800 mg, at least 1900 mg, at least 2000 mg, atleast 2100 mg, at least 2200 mg, at least 2300 mg, at least 2400 mg, atleast 2500 mg, at least 2600 mg, at least 2700 mg, at least 2800 mg, atleast 2900 mg, or at least 3000 mg, of nicotinamide riboside compound4). In some embodiments, the dose comprises at least 5 mg, at least 10,at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least60 mg, at least 80 mg, at least 100 mg, at least 120 mg, at least 140mg, at least 160 mg, at least 180 mg, at least 200 mg, at least 220 mg,at least 240 mg, at least 260 mg, at least 280 mg, at least 300 mg, atleast 320 mg, at least 340 mg, at least 360 mg, at least 380 mg, atleast 400 mg, at least 500 mg, at least 600 mg, at least 700 mg, atleast 800 mg, at least 900 mg, or at least 1000 mg of pterostilbene.

The compositions disclosed herein may be administered over any period oftime effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. The period of time may be at least 1 day, atleast 10 days, at least 20 days, at least 30, days, at least 60 days, atleast three months, at least six months, at least a year, at least threeyears, at least five years, or at least ten years. The dose may beadministered when needed, sporadically, or at regular intervals. Forexample, the dose may be administered monthly, weekly, biweekly,triweekly, once a day, or twice a day.

In some embodiments, the subject is given a test to measure the generalprogression or symptomatic progression of a motor neuron disease. Insome embodiments, the subject is given a motor function test and/or acognition and conduct function test. The motor function test may beRevised Amyotrophic Lateral Sclerosis Functional Rating Scale(ALSFRS-R). The cognition and conduct test may be Complutense VerbalLearning Test (TAVEC), Symbol Digit Modalities Test (SDMT), VerbalFluency Test, Digit Span (Wechsler Memory Scale III), D2 Attention Test,Wechsler Memory Scale III for Letters and Numbers, London Tower Test,Stroop test, Frontal System Behavior Scale (FrSBe), and/or Brief Test(subjective conduct). In some embodiments, subjects are given both motorfunction and cognitive function tests. Motor function or cognitivefunctions tests may be given to the subject once or multiple times.

In some embodiments, the method further comprises measuring a feature(e.g., a feature associated with inflammation) in the subject. In someembodiments, the feature is measured in a blood test. Examples offeatures that may be tested are the level of a cytokine, level ofamyloid A protein, level of macrophage activation marker neopterin,level of creatine phosphokinase (CPK), level of erythrocytesedimentation rate, level of C-reactive protein, plasma viscosity,and/or white blood cell count. In some embodiments, the cytokine isproinflammatory cytokine. In some embodiments, the cytokine is ananti-inflammatory cytokine. Examples of cytokines include, but are notlimited to, TNFα, IFNγ, IL-1, IL-6, IL-8, or TGFβ.

In some embodiments, the method further comprises administering a fattyacid supplement to the subject. In some embodiments, the fatty acidsupplement comprises an oil. The oil may be processed (e.g., refined,bleached, or deodorized). In other embodiments, the oil is unprocessedor a virgin oil. In some embodiments, the fatty acid supplement isderived or fractionated from a source to yield separated fatty acids. Insome embodiments, the oil is a coconut oil. Coconut oil, as used herein,may include any oil produced by the nut of the coconut palm. Fatty acidsfound in the supplements disclosed herein may be short-chain fattyacids, medium chain fatty acids, or long chain fatty acids. Exemplaryfatty acids that may be found in the supplement include, but are notlimited to, caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleicacid, linoleic acid, and/or linolenic acid. The fatty acid supplementdisclosed herein may comprise saturated fatty acids, unsaturated fattyacids, monounsaturated fatty acids, and/or polyunsaturated fatty acids.In some embodiments, the fatty acid supplement may comprise ahydrogenated oil. Fatty acid supplements may comprise one or more fattyacid(s). Actual dosage levels and administration regimen of the fattyacid supplement disclosed herein may be varied so as to obtain an amountof fatty acid supplement that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In further embodiments, the present invention provides methods relatingto:

improving fertility;

treating or preventing infertility;

inducing ovulation;

increasing sperm count; or

increasing lactation;

comprising administering to the subject Compound 4 prepared by thesynthetic methods disclosed herein.

In certain embodiments, such a method of treatment further comprisesadministering pterostilbene.

In some embodiments, improving fertility or treating and/or preventinginfertility comprises inducing ovulation and/or oocyte and folliclematuration in a female subject. In some embodiments, improving fertilityor treating and/or preventing infertility comprising increasing spermcount and/or sperm motility in a male subject.

In some embodiments, the subject is a mammal (e.g., a human, a non-humanmammal). The subject may be male or female. In some embodiments, thesubject has impaired fertility (e.g., reduced sperm count, reduced spermmotility, reduced ovulation, reduced follicle and/or oocyte maturation).In some embodiments, the subject is infertile or sterile.

In some embodiments, provided herein are methods of increasing overallsperm health and/or sperm count in a subject by administering to thesubject (e.g., a subject in need thereof) a composition disclosed herein(i.e., a composition comprising nicotinamide riboside (compound 4)and/or pterostilbene). Increasing sperm count may be achieved byincreasing the concentration of spermatozoa in seminal fluid, increasingthe absolute number of spermatozoa in semen and/or increasing the volumeof semen per ejaculate. The methods and compositions disclosed hereinmay increase overall sperm count by increasing or inducingspermatogenesis. In some embodiments, administering the compositionsdisclosed herein increase or improve sperm motility (e.g., increasingthe percentage of spermatozoa moving in semen or increasing the amountof time spermatozoa are moving) in a subject. In some embodiments,administering the compositions disclosed herein maintains or improvesoverall sperm health, sperm count, and/or sperm motility in the testesand/or epididymis post spermatogenesis.

In some embodiments, provided herein are methods of inducing and/orincreasing the likelihood of ovulation in a subject by administering tothe subject (e.g., a subject in need thereof) a composition disclosedherein (i.e., a composition comprising nicotinamide riboside (compound4) and/or pterostilbene). In some embodiments, provided herein aremethods of inducing follicle and oocyte maturation (e.g.,folliculogenesis) in a subject by administering to the subject (e.g., asubject in need thereof) a composition disclosed herein (i.e., acomposition comprising nicotinamide riboside (compound 4) and/orpterostilbene).

Disclosed herein are compositions and methods to aid in in vitrofertilization procedures and practices. In some aspects, provide hereinare methods of treating, preserving, or improving gamete (i.e., spermand/or oocyte) likelihood of fertilization in an in vitro procedure. Insome embodiments, compositions disclosed herein are added to semen(e.g., semen obtained from a donor subject) in preparation of forartificial insemination or intrauterine insemination (IUI). In someembodiments, compositions disclosed herein are added to semen inpreparation intracytoplasic sperm injection into an oocyte.

In some embodiments, the compositions disclosed herein may be used toenhance mitochondrial numbers, mitochondrial activity, cellular energylevels or cellular energy-producing potential in oocytes, postnatalfemale germline stem cells (also referred to herein as OSCs) and/orpreimplantation embryos prior to conducting and/or following methods ofin vitro fertilization. It has been recently discovered that adultfemale mammals retain rare germline or oogonial stem cells (OSCs) thatroutinely produce new oocytes in a manner analogous to germline stemcell support of sperm production in the adult testis, and these OSCs maybe new targets for in vitro fertilization therapies (Spradling, Nature2004 428:133-134). In some embodiments, provided herein are methods ofincreasing the overall viability of an oocyte removed from a subject(e.g., an oocyte removed in preparation for in vitro fertilization). Insome embodiments, an oocyte is treated or stored with a compositiondisclosed herein prior to in vitro fertilization. In one example,provided herein are methods of in vitro fertilization, the methodinvolving the steps of: incubating an oocyte from a subject with acomposition disclosed herein; and fertilizing the oocyte in vitro toform a zygote. In another example, the methods provided herein include amethod of in vitro fertilization, the method involving the steps of (a)incubating an OSC from a subject with composition disclosed herein; (b)obtaining a composition containing OSC mitochondria from the OSC; (c)transferring the composition into an isolated oocyte (e.g., an oocyteextracted from a subject); and (d) fertilizing the oocyte in vitro toform a zygote.

In some embodiments, the composition disclosed herein may be added to asolution used for in vitro fertilization procedures for oocytepreparation and/or storage, such as cell culture medium, oocyteretrieval solution, oocyte washing solution, oocyte in vitro maturationmedium, ovarian follicle in vitro maturation medium, oocyte in vitrofertilization medium, embryo culture medium, cleavage medium,vitrification solution, cryopreservation solution and/or embryo thawingmedium.

Gametes may be stored for any period of time with the compositionsdisclosed herein before in vitro fertilization is performed.

In some aspects, provided herein are methods of increasing lactation ina subject by administering to the subject (e.g., a subject in needthereof) a composition disclosed herein (i.e., a composition comprisingnicotinamide riboside (compound 4) and/or pterostilbene). In someembodiments, increasing lactation comprising increasing the rate atwhich milk is secreted and/or produced from the mammary glands of asubject. In some embodiments, increasing lactation comprises increasingthe volume of secreted milk in the subject. In some embodiments, thesubject is a human. In some embodiments the subject is a non-humananimal, such as a dairy animal (e.g., a cow, a buffalo, a goat, a sheep,a camel).

In some aspects, provided herein are methods of improving animalfecundity and/or breeding outcomes in animal husbandry by administeringthe compositions disclosed herein to a subject(s) (e.g., a non-humansubject). In some embodiments, increasing litter size in a subject(e.g., a non-human subject, such as a domesticated animal). In someembodiments, the subject is a mammal. The subject may be a rodent,lagomorph, feline, canine, porcine, ovine, bovine, equine, or primate.For example, a composition disclosed herein may be administered to afemale subject to increase number of offspring per litter orreproductive cycle. Alternately, the compositions disclosed herein maybe administered to male subjects to increase spermatozoa production toobtain semen samples with increased virility for use in artificialinsemination.

Actual dosage levels and administration regimen of the compositionsdisclosed herein may be varied so as to obtain an amount of nicotinamideriboside and/or pterostilbene that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In some embodiments, administration of the composition comprisesadministration of the composition in one or more dose(s). In someembodiments, administration of the composition comprises administrationof the composition in one or more, five or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, one hundred or more,or one thousand or more dose(s). In some embodiments, the dose comprisesat least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, atleast 125 mg, at least 150 mg, at least 200 mg, at least 225 mg, atleast 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, atleast 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, atleast 450 mg, at least 475 mg, at least 500 mg, at least 550 mg, atleast 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, atleast 800 mg, or at least 850 mg of nicotinamide riboside (compound 4).In some embodiments, the dose comprises at least 5 mg, at least 10 mg,at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg,at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least80 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 110 mg,at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, atleast 160 mg, least 170 mg, at least 180 mg, at least 190 mg, at least200 mg, or at least 250 mg of pterostilbene.

The compositions disclosed herein may be administered over any period oftime effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. The period of time may be at least 1 day, atleast 10 days, at least 20 days, at least 30, days, at least 60 days, atleast three months, at least six months, at least a year, at least threeyears, at least five years, or at least ten years. The dose may beadministered when needed, sporadically, or at regular intervals. Forexample, the dose may be administered monthly, weekly, biweekly,triweekly, once a day, or twice a day.

In further embodiments, the present invention provides methods relatingto:

treating or preventing kidney damage;

treating or preventing acute kidney injury;

treating or preventing kidney disease; or increasing blood flow to thekidneys;

comprising administering to the subject Compound 4 prepared by thesynthetic methods disclosed herein.

In certain embodiments, such a method of treatment further comprisesadministering pterostilbene.

In certain embodiments, the kidney damage is the result of cancer,decreased blood flood to the kidneys (i.e., ischemia), back up of urinein the kidneys, sepsis, trauma, autoimmune disease, drug-inducedtoxicity (e.g., non-steroidal anti-inflammatory (NSAID) inducednephrotoxicity), lead poisoning, and/or severe dehydration.

In some embodiments, the acute kidney injury is the result of decreasedblood flow to the kidneys (i.e., ischemic injury). Decreased blood flowto the kidneys may be a result of hypotension, blood loss, severediarrhea, heat attack, heart failure, deceased heart function, organfailure, drug-induced nephrotoxicity (e.g., NSAID inducednephrotoxicity), allergic reactions, burns, trauma (e.g., blunt trauma),and/or surgery. In some embodiments, the acute kidney injury is theresult of cancer (e.g., multiple myeloma), sepsis, vasculitis,interstitial nephritis, scleroderma, tubular necrosis,glomerulonephritis, or thrombotic microangiopathy. In some embodiments,the acute kidney injury is the result of blockage of the urinary tract.Blockage of the urinary tract may be caused by bladder cancer, prostatecancer, cervical cancer, an enlarged prostate, kidney stones, or bloodclots in the urine.

In some embodiments, the chronic kidney disease is the result of animmune system disease (e.g., lupus), long term viral disease (e.g.,HIV/AIDS, hepatitis B, or hepatitis C), urinary tract infections,polycystic kidney disease, and/or inflammation of glomeruli.

As used herein, kidney damage may refer to a medical condition ofimpaired kidney function in which the kidneys fail to adequately filtermetabolic wastes from the blood. In some embodiments, kidney damage isalso indicative of kidney failure. Examples of conditions that may causekidney damage include, but are not limited to decreased blood flood tothe kidneys, back up of urine in the kidneys, sepsis, trauma (e.g., suchas blunt trauma), an autoimmune disease, drug-induced nephrotoxicity(e.g., NSIAD induced nephrotoxicity), heavy mental poisoning (e.g., leadpoisoning), or severe dehydration.

In some aspects, provided herein are methods of treating or preventingacute kidney injury (i.e., in a subject in need thereof). In someembodiments, acute kidney injury is an episode of kidney failure orkidney damage that happens within a few hours or a few days. Acutekidney injury, as used herein, may be characterized by abruptdeterioration in kidney function. In some embodiments, the subject hasacute kidney injury, and the acute kidney injury may manifest by anincrease in serum creatinine level with or without reduced urine output.In some embodiments, the subject has increased serum creatinine levels.In some embodiments, the subject may have reduced urine output. In someembodiments, the subject has acute kidney injury, and the acute kidneyinjury may be prerenal (e.g., caused by decreased renal blood flow),intrinsic renal (e.g., caused by a process within the kidneys), orpostrenal (e.g., caused by inadequate drainage of urine distal to thekidneys). Acute kidney injury or kidney damage may be a result of use(e.g., overuse) of medications, such as NSAIDs, angiotensin-convertingenzyme inhibitors, angiotensin receptor blockers, cyclosporine,diuretics, tacrolimus, penicillin analogues, cephalosporins,sulfonamides, ciprofloxacin, acyclovir, rifampin, phenytoin, interferon,or proton pump inhibitors. Other causes of acute kidney injury andkidney damage include cardiorenal syndrome, hepatorenal syndrome,abdominal compartment syndrome, hypercalcemia, sepsis, neurogenic shock,infections of the renal parenchyma, glomerulonephritis, viral infections(such as Epstein-Barr virus infections or cytomegalovirus infections),bacterial infections (e.g., bacterial infections caused by bacteria ofthe Streptococcus or Legionella species), or fungal infections (e.g.,fungal infections caused by candidiasis or histoplasmosis). In someembodiments, the acute renal injury and/or kidney damage is caused by asystemic disease, such as sarcoidosis or lupus.

Additional examples of conditions that cause acute kidney injury andkidney damage include cancer (e.g., multiple myeloma), prolongedhypotension, renal vein thrombosis, malignant hypertension, sclerodermarenal crisis, renal atheroembolic disease, renal infarction vasculitis,interstitial nephritis, scleroderma, and or conditions that causeinflammation of or damage to the kidney tubules, such as tubularnecrosis, glomerulonephritis, or thrombotic microangiopathy.

In some embodiments, the acute kidney injury and/or kidney damage iscaused by a decrease in blood flow to the kidney. Conditions that maycause a decrease in blood flow to the kidneys includes, for example,blood loss, severe diarrhea, heat attack, heart failure, deceased heartfunction, organ failure, allergic reactions, burns, and/or trauma. Insome embodiments, the subject has undergone surgery, and the subject'sblood vessels have been clamped, leading to a decrease of blood flow tothe kidneys. In some embodiments, the acute kidney injury or kidneydamage is the result of a blockage of the urinary tract. A blockage ofthe urinary tract may be the result of, for example, neurogenic bladder,retroperitoneal fibrosis, bladder cancer, prostate cancer, cervicalcancer, an enlarged prostate, kidney stones, blood clots, or tumors.

In some embodiments, the subject has a kidney disease. A kidney diseaseis any condition that affects the kidney's ability to filter compoundsout of blood, filter extra water out of blood, and/or help control bloodpressure. Kidney disease may be caused by diabetes, hypertension, asystemic disease (e.g., lupus), viral disease (e.g., HIV/AIDS, hepatitisB, or hepatitis C), urinary tract infections, a genetic disease, such aspolycystic kidney disease, or any condition that results in theinflammation of kidney glomeruli.

In some embodiments, the subject's kidney function may be measuredprior, during or after administration of a composition disclosed herein.Kidney function may be evaluated as a function of glomerular filtrationrate, urine output, or the level of other biomedical markers of kidneyhealth, such as creatinine, urea, nitrogen, phosphorus, or potassium.Markers such as creatinine, urea, nitrogen, phosphorus, or potassium maybe measured in the urine or through a blood test.

Actual dosage levels and administration regimen of the compositionsdisclosed herein may be varied so as to obtain an amount of nicotinamideriboside (compound 4) and/or pterostilbene that is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

In some embodiments, administration of the composition comprisesadministration of the composition in one or more dose(s). In someembodiments, administration of the composition comprises administrationof the composition in one or more, five or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, one hundred or more,or one thousand or more dose(s). In some embodiments, the dose comprisesat least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, atleast 125 mg, at least 150 mg, at least 200 mg, at least 225 mg, atleast 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, atleast 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, atleast 450 mg, at least 475 mg, at least 500 mg, at least 550 mg, atleast 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, atleast 800 mg, or at least 850 mg of nicotinamide riboside (Compound 4).In some embodiments, the dose comprises at least 5 mg, at least 10 mg,at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg,at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least80 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 110 mg,at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, atleast 160 mg, least 170 mg, at least 180 mg, at least 190 mg, at least200 mg, or at least 250 mg of pterostilbene.

The compositions disclosed herein may be administered over any period oftime effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. The period of time may be at least 1 day, atleast 10 days, at least 20 days, at least 30, days, at least 60 days, atleast three months, at least six months, at least a year, at least threeyears, at least five years, or at least ten years. The dose may beadministered when needed, sporadically, or at regular intervals. Forexample, the dose may be administered monthly, weekly, biweekly,triweekly, once a day, or twice a day.

In further embodiments, the present invention provides methods relatingto treating and/or preventing liver related diseases and disorders andfor improving liver health in a subject by administering to the subjectCompound 4, as prepared by the synthetic methods disclosed herein.Specifically, the invention provides a method of:

treating or preventing liver damage;

treating or preventing fatty liver;

decreasing the serum level of alanine transaminase (ALT); or decreasingthe serum level of aspartate transaminase (AST) in a subject;

comprising administering to the subject Compound 4 prepared by thesynthetic methods disclosed herein.

In certain embodiments, such a method of treatment further comprisesadministering pterostilbene.

In certain embodiments, the liver damage is the result of cancer (e.g.,liver cancer, bile duct cancer and/or a liver adenoma), cirrhosis, viralinfection (e.g., hepatitis A infection, a hepatitis B infection and/or ahepatitis E infection), congenital disorders of metabolism, trauma,autoimmune disease (e.g., autoimmune hepatitis, primary biliarycirrhosis, or primary sclerosing cholangitis), hemochromatosis,hyperoxaluria, oxalosis, Wilson's disease and/or drug-inducedhepatotoxicity (e.g., alcohol-induced hepatotoxicity and/oracetaminophen-induced hepatotoxicity).

Provided herein are methods of preventing or treating liver damageand/or fatty liver in a subject by administering to the subject (e.g., asubject in need thereof) a composition disclosed herein (i.e., acomposition comprising nicotinamide riboside and/or pterostilbene).

In some embodiments, the subject may have or be predisposed to liverdamage and/or fatty liver. Liver damage may result from any conditionthat causes the cells of the liver (i.e., hepatocytes) to die orotherwise not function normally. Examples of conditions that may causeliver damage include, but are not limited to, cancer (e.g., livercancer, bile duct cancer, or a liver adenoma), trauma, congenitalmetabolic disorders (e.g., genetic metabolic disorders resulting in anenzyme deficiency), vascular injury, cirrhosis, a viral infection (e.g.,hepatitis A, hepatitis B, hepatitis E), an autoimmune disease (e.g.,autoimmune hepatitis, primary biliary cirrhosis, or primary sclerosingcholangitis), hemochromatosis, hyperoxaluria, oxalosis, Wilson'sdisease, or drug-induced hepatotoxicity (e.g., alcohol-inducedhepatotoxicity or acetaminophen-induced hepatotoxicity) Fatty liver maybe caused by any condition that causes fat accumulation of liver. Theseconditions may be, but are not limited to, non-alcoholic fatty liverdisease or alcoholic liver disease.

Disclosed herein are methods of treating or preventing age-relatedsymptoms or diseases comprising administering a composition disclosedherein. Provided herein are methods of decreasing the amount of alaninetransaminase (ALT) and/or aspartate transaminase (AST) in a subjectcomprising administering to the subject a composition provided herein.AST and ALT are reasonably sensitive indicators of liver damage orinjury from different types of diseases or conditions, and they areoften measured in liver tests or liver blood tests. Elevated levels ofAST and ALT are associated with liver damage and liver malfunction. Insome embodiments, ALT is decreased in the subject by at least 0.1 U/L,at least 0.2 U/L, at least 0.3 U/L, at least 0.4 U/L, at least 0.5 U/L,at least 0.6 U/L, at least 0.7 U/L, at least 0.8 U/L, at least 0.9 U/L,at least 01.0 U/L, 1.1 U/L, at least 1.2 U/L, at least 1.3 U/L, at least1.4 U/L, at least 1.5 U/L, at least 1.6 U/L, at least 1.7 U/L, at least1.8 U/L, at least 1.9 U/L, at least 2.0 U/L, 2.1 U/L, at least 2.2 U/L,at least 2.3 U/L, at least 2.4 U/L, at least 2.5 U/L, at least 2.6 U/L,at least 2.7 U/L, at least 2.8 U/L, at least 2.9 U/L, at least 3.0 U/L,at least 3.5 U/L, 4.0 U/L, at least 4.5 U/L, or at least 5.0 U/L afteradministration of the composition. In some embodiments, the ALT isdecreased by at least 0.1 U/L, at least 0.2 U/L, at least 0.3 U/L, atleast 0.4 U/L, at least 0.5 U/L, at least 0.6 U/L, at least 0.7 U/L, atleast 0.8 U/L, at least 0.9 U/L, at least 01.0 U/L, 1.1 U/L, at least1.2 U/L, at least 1.3 U/L, at least 1.4 U/L, at least 1.5 U/L, at least1.6 U/L, at least 1.7 U/L, at least 1.8 U/L, at least 1.9 U/L, at least2.0 U/L, 2.1 U/L, at least 2.2 U/L, at least 2.3 U/L, at least 2.4 U/L,at least 2.5 U/L, at least 2.6 U/L, at least 2.7 U/L, at least 2.8 U/L,at least 2.9 U/L, at least 3.0 U/L, at least 3.5 U/L, 4.0 U/L, at least4.5 U/L, or at least 5.0 U/L after administration of the composition.

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme that participatesin many metabolic reactions. NAD+ plays an important role intranscription regulation, longevity, and age-associated diseases. NAD+levels decrease with age, while increased NAD+ levels are associatedwith robust health. In some embodiments, provided herein are methods ofincreasing the amount of NAD+ of a subject by administering acomposition disclosed herein. NAD+ may increase by at least 1.0 μg/mL,at least 2.0 μg/mL, at least 3.0 μg/mL, at least 4.0 μg/mL, at least 5.0μg/mL, at least 6.0 μg/mL, at least 7.0 μg/mL, at least 8.0 μg/mL, atleast 9.0 μg/mL, at least 10.0 μg/mL, at least 11.0 μg/mL, at least 12.0μg/mL, at least 13.0 μg/mL, at least 14.0 μg/mL, at least 15.0 μg/mL, atleast 16 μg/mL, at least 17 μg/mL, at least 18 μg/mL, at least 19 μg/mL,at least 20 μg/mL, at least 21 μg/mL, at least 22 μg/mL, at least 23μg/mL, at least 24 μg/mL, at least 25 μg/mL, at least 26 μg/mL, at least27 μg/mL, at least 28 μg/mL, at least 29 μg/mL, or at least 30 μg/mLafter administration of the composition.

Provided herein are methods of decreasing blood pressure (e.g.,diastolic blood pressure) of a subject by administering a compositionherein. In some embodiments, the subject's diastolic blood pressuredecreases by at least 1 mmHg, at least 1.5 mmHg, at least 2 mmHg, atleast 2.5 mmHg, at least 3 mmHg, at least 3.5 mmHg, at least 4.0 mmHg,at least 4.5 mmHg, or at least 5 mmHg after administration of thecomposition.

In certain embodiments, the invention also provides a method of treatinga disease comprising administering to a subject compound 4 and/orpterostilbene.

In certain such embodiments, the disease is a neurodegenerative disease.Exemplary neurodegenerative diseases include Alzheimer's, Parkinson's,and Huntington's Disease.

In alternative such embodiments, the disease is a skin disorder. Skindisorders may be caused by exposure to the sun; exemplary such disordersare selected from the group consisting of actinic keratoses, lentiginesor age spots, seborrheic keratoses, sun burn, photosensitivity, moles,polymorphous light eruption, solar elastosis or wrinkles, skin cancer(such as melanoma, squamous cell carcinoma, and basal cell carcinoma),and freckles. Skin disorders may also be caused by inflammation;exemplary such disorders are selected from the group consisting ofpsoriasis, contact dermatitis, atopic dermatitis, seborrheic dermatitis,asteatotic eczema, discoid eczema, hand eczema, gravitational/varicoseeczema, eczematous drug eruptions, lichen simplex, acne, lichen planus,pityriasis lichenoides, keratosis lichenoides chronica, lichen nitidus,lichen striatus, mycosis fungoides, erythroderma, erythema multiforme,Stevens-Johnson Syndrome, vasculitis, and toxic epidermal necrolysis. Infurther embodiments, a skin disorder may be caused by autoimmune diseaseis selected from the group consisting of pyoderma gangrenosum, systemiclupus erythematosus, eosinophilic fasciitis, scleroderma, pemphigusvulgaris, bullous pemphigoid, alopecia areata, vitiligo, psoriasis,dermatomyositis, and dystrophic epidermolysis bullosa.

In yet alternative embodiments, the disease is obesity or diabetes. Themethods may also enable lipid lowering and blood pressure reduction.

In further alternative embodiments, the disease is muscle wasting orsarcopenia.

In further alternative embodiments, the disease is an autoimmune diseasesuch as arthritis or lupus.

In further alternative embodiments, the disease is mitochondrialdysfunction or disease.

In still further alternative embodiments, the disease is an acceleratedaging disease such as progeria.

In other embodiments, the disease is chemotherapy induced neuropathy, orcognitive decline. The methods may also enable pain reduction.

In yet further embodiments, the disease is a cancer such as breastcancer, colon cancer, etc.

In other embodiments, the invention provides methods that promotesirtuin activation, NAD+ boosting, PARP activation, autophagy andmitophagy, or mitochondrial biogenesis.

In still further embodiments, the invention provides methods ofpromoting wellness comprising administering to a subject compound 4and/or pterostilbene.

In some embodiments, the methods promote wellness by promoting stem cellhealth and function; such stem cells may be intestinal, skin, muscle,hematopoietic, or neural stem cells.

The methods may also promote wellness by preventing hearing loss or hairloss.

Alternatives, the methods may promote healthy hair and nail growth.

In yet further alternative embodiments, the methods may promote musclebuilding for endurance and strength, weight loss, or and increasedimmune response (innate and adaptive function).

Actual dosage levels and administration regimen of the compositionsdisclosed herein may be varied so as to obtain an amount of nicotinamideriboside (compound 4) and/or pterostilbene that is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

In some embodiments, administration of the composition comprisesadministration of the composition in one or more dose(s). In someembodiments, administration of the composition comprises administrationof the composition in one or more, five or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, one hundred or more,or one thousand or more dose(s). In some embodiments, the dose comprisesat least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, atleast 125 mg, at least 150 mg, at least 200 mg, at least 225 mg, atleast 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, atleast 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, atleast 450 mg, at least 475 mg, at least 500 mg, at least 550 mg, atleast 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, atleast 800 mg, or at least 850 mg of nicotinamide riboside (compound 4).In some embodiments, the dose comprises at least 5 mg, at least 10 mg,at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg,at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least80 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 110 mg,at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, atleast 160 mg, least 170 mg, at least 180 mg, at least 190 mg, at least200 mg, or at least 250 mg of pterostilbene.

The compositions disclosed herein may be administered over any period oftime effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. The period of time may be at least 1 day, atleast 10 days, at least 20 days, at least 30, days, at least 60 days, atleast three months, at least six months, at least a year, at least threeyears, at least five years, or at least ten years. The dose may beadministered when needed, sporadically, or at regular intervals. Forexample, the dose may be administered monthly, weekly, biweekly,triweekly, once a day, or twice a day.

In any of the treatment methods described herein, compound 4, alone orin combination with pterostilbene, may be formulated together with oneor more pharmaceutically acceptable carriers (additives) and/ordiluents. In another aspect, the agents described herein can beadministered as such, or administered in mixtures with pharmaceuticallyacceptable carriers and can also be administered in conjunction withother agents. Conjunctive therapy thus includes sequential, simultaneousand separate, or co-administration of one or more compounds of theinvention, wherein the therapeutic effects of the first administered hasnot entirely disappeared when the subsequent compound is administered.

Compositions useful for the methods of the invention are describedabove.

EXEMPLIFICATION

The invention described generally herein will be more readily understoodby reference to the following examples, which are included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and are not intended to limit the invention.

Example 1. Exemplary Syntheses of Compound 3

A panel of experiments was conducted wherein ribofuranose tetraacetate(compound 1) was treated with a chlorinating agent to form compound 2,followed by treatment with nicotinamide to form compound 3. Results ofthe panel are summarized in the table below.

Volume Weight (g) (mL) mol equivalents Experiment 1A Ribofuranosetetraacetate 20.0 HCl 4.00 Dioxane 2.00 Dichloromethane 100 5.00Nicotinamide 1.00 Acetonitrile 100 6.00 Procedure: HCl in dioxane wasadded to ribofuranose tetraacetate in DCM for 12 hours at 25- 30° C..Crude was isolated and dissolved in acetonitrile. Nicotinamide was addedand stirred for 16 hours at 25-30° C.. Added tributylamine and stirredfor 30 minutes then rinsed with acetone. Off-white product was formedwith 52% yield. Experiment 1B Ribofuranose tetraacetate 20.0 HCl 4.00Dioxane 2.00 Acetyl chloride Dichloromethane 100 5.00 Nicotinamide 1.00Acetonitrile 100 6.00 Procedure: HCl in dioxane was added toribofuranose tetraacetate in DCM for 12 hours at 25- 30° C.. Crude wasisolated and dissolved in acetonitrile. Nicotinamide was added andstirred for 16 hours at 25-30° C.. Added tributylamine and stirred for30 minutes then rinsed with acetone. Off-white product was formed with54% yield. Experiment 1C Ribofuranose tetraacetate 50.0 0.157 Thionylchloride 60.0 5.20 Dichloromethane 300 6.00 Acetic acid 25.0 0.500Nicotinamide 19.1 1.00 Acetonitrile 300 6.00 Tributylamine 10.0 0.200Procedure: Thionyl chloride was added to ribofuranose tetraacetate andacetic acid in DCM for 16 hours at 25-30° C.. Crude was isolated anddissolved in acetonitrile. Nicotinamide was added and stirred for 16hours at 25-30° C.. Reaction was cooled to 0-5° C. and tributylamine wasadded for 15 minutes and stirred for 30 minutes. Off-white product wasformed with 42% yield. Experiment 1D Ribofuranose tetraacetate 100 0.314HCl 4.00 Dioxane 150 2.00 Dichloromethane 600 6.00 Nicotinamide 42.21.10 Acetonitrile 600 6.00 Procedure: HCl in dioxane was added toribofuranose tetraacetate in DCM for 12 hours at 25- 30° C.. Crude wasisolated and dissolved in acetonitrile. Nicotinamide was added andstirred for 16 hours at 25-30° C.. Experiment 1E Ribofuranosetetraacetate 200 0.628 HCl 4.00 Dioxane 393 2.50 Acetyl chloride 22.50.500 Dichloromethane 1000 6.00 Nicotinamide 84.6 1.10 Acetonitrile 12006.00 Procedure: HCl in dioxane was added to ribofuranose tetraacetate inDCM for 12 hours at 25- 30° C.. Crude was isolated and dissolved inacetonitrile. Nicotinamide was added and stirred for 16 hours at 25-30°C.. Reaction was cooled to 0-5° C. and tributylamine was added stirredfor 30 minutes then rinsed with acetonitrile and acetone. Product wasformed with 59% yield. Experiment 1F Ribofuranose tetraacetate 500 1.57HCl 4.00 Dioxane 1570 4.00 Dichloromethane 3000 6.00 Nicotinamide 1921.00 Acetonitrile 3000 6.00 Tributylamine 100 0.200 Procedure: HCl indioxane was added to ribofuranose tetraacetate in DCM for 16 hours at25- 30° C.. Crude was isolated and dissolved in acetonitrile.Nicotinamide was added and stirred for 16 hours at 25-30° C.. Reactionwas cooled to 0-5° C. and tributylamine was added stirred for 30 minutesthen rinsed with acetonitrile and acetone. Product was formed with 51%yield. Experiment 1G Ribofuranose tetraacetate 4000 HCl 4.00 Dioxane8000 2.00 Dichloromethane 14000 3.50 Acetyl chloride 400 0.100Nicotinamide 1690 0.420 Acetonitrile 14000 3.50 Tributylamine 800 0.200Procedure: HCl in dioxane was added to ribofuranose tetraacetate in DCMand acetyl chloride for 16 hours at 25-30° C.. Crude was isolated anddissolved in acetonitrile. Nicotinamide was added and stirred for 16hours at 25-30° C.. Reaction was cooled to 0-5° C. and tributylamine wasadded stirred for 30 minutes then rinsed with acetonitrile and acetone.Product was formed with 63% yield. Experiment 1H Ribofuranosetetraacetate 1000 3.14 HCl 4.00 Dioxane 3140 4.00 Dichloromethane 60006.00 Nicotinamide 384 1.00 Acetonitrile 6000 6.00 Tributylamine 2000.200 Procedure: HCl in dioxane was added to ribofuranose tetraacetatein DCM for 16 hours at 25- 30° C.. Crude was isolated and dissolved inacetonitrile. Nicotinamide was added and stirred for 16 hours at 25-30°C.. Reaction was cooled to 0-5° C. and tributylamine was added stirredfor 30 minutes then rinsed with acetonitrile and acetone. Product wasformed with 51% yield.

From the panel of experiments outlined above, the following points wereincorporated into the larger scale process of converting compound 1 tocompound 3:

Molar equivalents of nicotinamide are minimized in the range of 1.0 to1.1 as the nicotinamide hydrochloride (byproduct) is difficult toremove.

Molar equivalents of HCl are minimized at 1.0 in order to reduce thedistillation time. Experiments demonstrated that chloro-intermediatecompound 2 is not stable.

Acetyl chloride can be added as a drying agent.

Generally, thionyl chloride was less reactive as compared to HCl and hasthe disadvantage of generating sulfur containing byproducts.

Example 2. Scale-Up Syntheses of Compound 3

246 kg of 4M HCl in dioxane was added to 294 kg of ribofuranosetetraacetate in 471 kg of acetonitrile and stirred for 16 hours at10-20° C. A sample for reaction completion was taken with aspecification of NMT 5% ribofuranose tetraacetate. 237 kg ofacetonitrile and 127 kg of nicotinamide were added and stirred for 20hours at 25-30° C. Reaction was cooled to 0-10° C. and 51 kg oftributylamine was added and stirred for 60 minutes. Product was isolatedvia filtration and rinsed with 177 kg of acetonitrile and then 474 kg ofacetone. Product was dried in vacuum at less than 30 in Hg and NMT 40°C. Product was formed with 49% yield.

Example 3: Exemplary Syntheses of Compound 4

Volume Weight (g) (mL) mol equivalents Experiment 3A Nicotinamideriboside triacetate 5.00 0.0120 Methanol 25.0 5.00 3M Methanolic NH₃20.0 5.00 Procedure: Methanolic NH₃ is added to nicotinamide ribosidetriacetate and kept overnight at −15 to −10° C.. Mizxture was cooled to−5 to 0° C. and pH adjusted to 6-7 with HCl. Meth- anol was added to thefiltrates and co-distilled. Brownish product was formed with 54% yield.Experiment 3B Nicotinamide riboside triacetate 150 0.360 Methanol 7505.00 4M Methanolic NH₃ 360 4.00 Procedure: Methanolic NH₃ is added tonicotinamide riboside triacetate and kept overnight at −15 to −10° C..Methanol was added to the crude and co-distilled. Methanol was added (2vol.) and pH was adjusted to 4-5 with HCl in methanol. The methyl tertbutyl ether (MTBE) (1 vol.) was added and stirred for 30 minutes at roomtemperature. Light brown product was formed with 82% yield. Experiment3C Nicotinamide riboside triacetate 100 0.240 Methanol 500 5.00 4MMethanolic NH₃ 240 4.00 Procedure: Methanolic NH₃ is added tonicotinamide riboside triacetate and kept overnight at −15 to −10° C..Methanol was added to the crude and co-distilled. Methanol was added (2vol.) and pH was adjusted to 4-5 with HCl in methanol and stirred for 1hour at room temperature. Light brown product was formed with 77% yield.Experiment 3D Nicotinamide riboside triacetate 100 0.240 Methanol 5005.00 4M Methanolic NH₃ 240 4.00 Procedure: Methanolic NH₃ was added tonicotinamide riboside triacetate and kept overnight at −15 to −10° C..Methanol was added and pH was adjusted to 4-5 with HCl in methanol at 0-10° C.. Methanol was added to the crude and co-distilled twice. Methanolwas added (2 vol.) and stirred for 1 hour at room temperature. Lightbrown product was formed with 78% yield. Experiment 3E Nicotinamideriboside triacetate 100 0.240 Methanol 500 5.00 4M Methanolic NH₃ 2404.00 Procedure: Methanolic NH₃ was added to nicotinamide ribosidetriacetate and kept overnight at −15 to −10° C.. pH was adjusted to 4-5with sulfuric acid at 0-10° C.. Methanol was added to the crude andco-distilled twice. Methanol was added (2 vol.) and stirred for 1 hourat room temperature. Light brown product was formed with 79% yield.Experiment 3F Nicotinamide riboside triacetate 1000 2.40 Methanol 50005.00 7M Methanolic NH₃ 1700 5.00 Procedure: Methanolic NH₃ was added tonicotinamide riboside triacetate and kept for 16 hour at −10 to −5° C..Methanol was added to the crude and co-distilled. Solids were filteredand washed with methanol and acetone. Off white product was formed with68% yield.

Example 4. Scale-Up Syntheses of Compound 4

408 kg of 7M methanolic NH₃ was added to 300 kg of nicotinamide ribosidetriacetate in 891 kg of methanol and stirred for 15 hour at −10 to 0° C.A sample for reaction completion was taken with a specification of NMT5% nicotinamide riboside triacetate. Distillation was completed for NLT15 hours with vacuum at less than 30 in Hg and temperature NMT 20° C.Distillation continued until at least 300 kg of distillate wascollected. Product was isolated via filtration and rinsed with 475 kg ofacetone and then 444 kg of MTBE. Product was dried in vacuum at lessthan 30 in Hg and NMT 40° C. Product was formed with 55% yield.

Example 5. Alternative Synthesis of Compound 3

Step 1: Chlorination

600 g D-Ribofuranose Tetraacetate was charged to a 5 L reactor followedby an acetonitrile rinse (942 g) at 22° C. The mixture was agitated andthe D-Ribofuranose Tetraacetate dissolved, thereby forming a clearsolution. An endotherm to 6.9° C. was observed. The reactor contentswere cooled to 0-5° C., then HCl gas (75.6 g, 1.1 equiv) was chargedsubsurface over 12 hours in ˜8 g doses every hour. An IPC sample forreaction completion indicated 8.9 A % D-Ribofuranose Tetraacetate (IPCspecification: not more than 5 A % Tetraacetate via CAD Detector). Thus,a supplemental charge of HCl gas (65.8 g, 0.96 equiv) was charged over 2h in 2 doses. A second IPC sample for reaction completion indicated 2.3A % D-Ribofuranose Tetraacetate. The reactor jacket temperature wasadjusted to 15° C.

Step 2: Nicotinamide Addition

Nicotinamide (253.8 g; 1.1 equiv) was charged to the reactor, resultingin a white suspension which exothermed to 26° C. Acetonitrile (474.0 g)was added to the reactor. The reactor contents were adjusted to 15° C.and tributylamine (384.4 g, 1.1 equiv) was charged over not less than 20min followed by an acetonitrile rinse (57 g). A moderate exotherm wasobserved (max 24° C.). The reactor contents were adjusted to 25° C. andagitated for 18 h. An IPC sample for reaction completion indicated 38 A% Nicotinamide (IPC specification: not more than 50% Nicotinamide at 260nm). No change was seen after 31 h. a. Nicotinamide Riboside ChlorideTriacetate (NR-A) to Anomer Ratio: 70.3%: 29.7% b. In situ YieldAnalysis of NR-A: 59.8% (469.9 g)

Step 3: Supplemental Tributylamine Charge (Due to Overcharge of HCl Gas)

Prior to filtration, a 100-200 mg aliquot of the slurry was filtered andwashed with acetone and analyzed to show 21.9 A % residual nicotinamide(from acetone-insoluble nicotinamide HCl). Accordingly, the productslurry was cooled to 10° C. and a supplemental charge of tributylamine(45.8 g, 0.131 equiv) was added to compensate for the charge of HCl gas(2.1 equiv). The slurry was warmed to 25° C., held for 2 h, the cooledto 0° C. and allowed to stir overnight (13 h). Prior to filtration a100-200 mg aliquot of the slurry was filtered and washed with acetoneand analyzed to show 1.8 A % residual nicotinamide.

Step 4: Isolation of Nicotinamide Riboside Chloride Triacetate (NR-A)

The pale yellow slurry was chilled to 0° C. (0 to 5° C.) and vacuumfiltered through centrifuge cloth (20-25 μm pore size). Acetone (1200 gor 948 g) was charged to the empty reactor and cooled to 0-1° C. withstirring to mobilize product residue. After complete deliquoring in thefilter, the product cake was washed with the chilled acetone. Theproduct was dried under vacuum for 24 h at 40-45° C. to afford NR-A(378.65 g (48.2% yield)) as white chalky solids (2.0 A % Nicotinamide,97.4 A % NR-A (260 nm), 0.13 A % Anomer). Product was 100 A % via CADDetector. Moisture Balance: 0.26% wt loss (75° C., 30 min).

Example 6. Alternative Synthesis of Compound 4

Step 1: Hydrolysis of Acetate Groups

A 5 L jacketed reactor was charged with methanol (350 g). NR-A (350 g)was charged to reactor at 22° C. to give a stirred suspension followedby methanol (350 g) rinse. The reactor contents were cooled to below −5to −3° C. and diethylamine (49.35 g, 0.8 equiv) was charged to thesuspension maintaining not more than 1° C. followed by methanol (133 g)rinse. The reaction mixture was stirred at −3° C. for not less than 2hours and the suspension became a tea colored solution (at plant-scale apolish filtration may be performed). The reaction was left to stir for19 h at −3° C. An IPC sample taken at 19 h for reaction completionindicated 4.4% monoacetate (IPC specification: not more than 5%monoacetate at 270 nm). After 28 h: 1.5% monoacetate. An aqueous 3N HCl(0.76 equiv) solution was prepared by adding 37% HCl (63.0 g, 0.76equiv) to water (189 g). The 3N HCl (0.76 equiv) quench solution wasadded to the reactor maintaining not more than 2° C. (1 h addition).During the 3N HCl addition, precipitation commenced. The mixture wasaged at −3° C. to 0° C. for not less than 12 hours.

Step 2: Isolation of Nicotinamide Riboside Chloride (NR-B)

The resulting thick pale yellow suspension was vacuum filtered throughcentrifuge cloth (20-25 μm pore size) at not more than 0-5° C. EthanolSDA-3C was charged to the reactor (553 g, 700 mL) and cooled to 0-5° C.The contents were used to rinse the product cake. Acetone (549 g, 700mL) was charged to the reactor and the contents were cooled to below 0°C. The contents were used to rinse the product cake. MTBE (518.0 g, 700mL) was charged to the reactor and the contents were cooled to below 0°C. The contents were used to rinse the product cake. The product wasdried under vacuum for 24 h at 30-40° C. to afford NR-B (126.9 g (52%yield)) as a pale yellow powder (0.3 A % Nicotinamide, 99.7 A % NR-B(270 nm), and 98.7 wt % vs working lab standard. Moisture Balance: 0.56%wt loss (75° C., 30 min). Percent product loss to filtrate: 43.7 g NR-B,17.9% of theory.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

What is claimed is:
 1. A method of preparing compound 3,

comprising the steps of: (a) combining compound 1 and a chlorinatingagent, thereby producing a first reaction mixture comprising compound 2,wherein: compound 1 is represented by

compound 2 is represented by

 and the chlorinating agent is gaseous HCl or HCl in dioxane; and (b)combining compound 2 with nicotinamide, thereby producing a secondreaction mixture comprising compound
 3. 2. The method of claim 1,wherein the chlorinating agent is gaseous HCl.
 3. The method of claim 1,wherein the chlorinating agent is HCl in dioxane.
 4. The method of anyone of claims 1-3, wherein step (a) occurs in a first solvent.
 5. Themethod of claim 4, wherein the first solvent is acetonitrile and thechlorinating agent is gaseous HCl.
 6. The method of claim 4, wherein thefirst solvent is dioxane and the chlorinating agent is HCl in dioxane.7. The method of claim 4, wherein the first solvent is dichloromethaneand the chlorinating agent is HCl in dioxane.
 8. The method of any oneof claims 3, 4, 6, and 7, wherein step (a) further comprises acetylchloride.
 9. The method of any one of claims 1-8, further comprisingstep (a′), which occurs after step (a) and before step (b): (a′)concentrating the first reaction mixture under reduced atmosphericpressure.
 10. The method of any one of claims 1-9, further comprisingcombining compound 2 and a second solvent.
 11. The method of claim 10,wherein the second solvent is acetonitrile.
 12. The method of any one ofclaims 1-9, wherein step (b) occurs in a second solvent.
 13. The methodof claim 12, wherein the second solvent is acetonitrile.
 14. The methodof any one of claims 1-13, further comprising step (b′), which occursafter step (b): (b′) combining the second reaction mixture with atrialkylamine.
 15. The method of claim 14, wherein the trialkylamine istributylamine.
 16. The method of any one of claims 1-15, furthercomprising: (c) combining compound 3 with an ammonia reagent, therebyproducing a third reaction mixture comprising compound 4, wherein:compound 4 is represented by

 and the ammonia reagent is methanolic ammonia or diethylamine.
 17. Themethod of claim 16, wherein the ammonia reagent is diethylamine.
 18. Themethod of claim 16, wherein the ammonia reagent is methanolic ammonia.19. The method of claim 18, wherein the methanolic ammonia is 7Mmethanolic ammonia.
 20. The method of any one of claims 16-19, whereinstep (c) occurs in a third solvent.
 21. The method of claim 20, whereinthe third solvent is methanol.
 22. The method of any one of claims16-21, wherein step (c) takes place at a temperature below roomtemperature.
 23. The method of any one of claims 16, 17, and 20-22,wherein the ammonia reagent is diethylamine; further comprising step(c′), which occurs after step (c): (c′) adding cold aqueous HCl to thethird reaction mixture, thereby producing a fourth mixture comprisingcompound
 4. 24. The method of any one of claims 16 and 18-22, whereinthe ammonia reagent is methanolic ammonia; further comprising step (c′),which occurs after step (c): (c′) concentrating the third reactionmixture under reduced atmospheric pressure, thereby producing a fourthmixture comprising compound
 4. 25. The method of claim 23 or 24, furthercomprising step (c″), which occurs after step (c′): (c″) filtering thefourth mixture.
 26. The method of claim 25, further comprising rinsingthe retentate with cold methanol or ethanol.
 27. The method of claim 25or 26, further comprising rinsing the retentate with cold acetone.
 28. Amethod of preparing compound 4,

comprising the steps of: (c) combining compound 3 with an ammoniareagent, thereby producing a first reaction mixture comprising compound4, wherein: compound 3 is represented by

 and the ammonia reagent is methanolic ammonia or diethylamine.
 29. Themethod of claim 28, wherein the ammonia reagent is diethylamine.
 30. Themethod of claim 28, wherein the ammonia reagent is methanolic ammonia.31. The method of claim 30, wherein the methanolic ammonia is 7Mmethanolic ammonia.
 32. The method of any one of claims 28-31, whereinstep (c) occurs in a third solvent.
 33. The method of claim 32, whereinthe third solvent is methanol.
 34. The method of any one of claims28-33, wherein step (c) takes place at a temperature below roomtemperature.
 35. The method of any one of claims 28, 29, and 32-34,wherein the ammonia reagent is diethylamine; further comprising step(c′), which occurs after step (c): (c′) adding cold aqueous HCl to thethird reaction mixture, thereby producing a fourth mixture comprisingcompound
 4. 36. The method of any one of claims 28 and 30-34, whereinthe ammonia reagent is methanolic ammonia; further comprising step (c′),which occurs after step (c): (c′) concentrating the third reactionmixture under reduced atmospheric pressure, thereby producing a fourthmixture comprising compound
 4. 37. The method of claim 35 or 36, furthercomprising step (c″), which occurs after step (c′): (c″) filtering thefourth mixture.
 38. The method of claim 37, further comprising rinsingthe retentate with cold methanol or ethanol.
 39. The method of claim 37or 38, further comprising rinsing the retentate with cold acetone. 40.The method of any one of claims 28-39, further comprising (a) combiningcompound 1 and a chlorinating agent, thereby producing a first reactionmixture comprising compound 2, wherein: compound 1 is represented by

compound 2 is represented by

 and the chlorinating agent is gaseous HCl or HCl in dioxane; and (b)combining compound 2 with nicotinamide, thereby producing a secondreaction mixture comprising compound
 3. 41. The method of claim 40,wherein the chlorinating agent is gaseous HCl.
 42. The method of claim40 wherein the chlorinating agent is HCl in dioxane.
 43. The method ofany one of claims 40-42, wherein step (a) occurs in a first solvent. 44.The method of claim 43, wherein the first solvent is acetonitrile andthe chlorinating agent is gaseous HCl.
 45. The method of claim 43,wherein the first solvent is dioxane and the chlorinating agent is HClin dioxane.
 46. The method of claim 43, wherein the first solvent isdichloromethane and the chlorinating agent is HCl in dioxane.
 47. Themethod of any one of claims 42, 43, 45, and 46, wherein step (a) furthercomprises acetyl chloride.
 48. The method of any one of claims 40-47,further comprising step (a′), which occurs after step (a) and beforestep (b): (a′) concentrating the first reaction mixture under reducedatmospheric pressure.
 49. The method of any one of claims 40-48, furthercomprising combining compound 2 and a second solvent.
 50. The method ofclaim 49, wherein the second solvent is acetonitrile.
 51. The method ofany one of claims 40-48, wherein step (b) occurs in a second solvent.52. The method of claim 51, wherein the second solvent is acetonitrile.53. The method of any one of claims 40-52, further comprising step (b′),which occurs after step (b): (b′) combining the second reaction mixturewith a trialkylamine.
 54. The method of claim 53, wherein thetrialkylamine is tributylamine.
 55. Compound 4, made according to methodof any one of claims 16-54.
 56. The compound of claim 55, whereincompound 4 is produced in a yield of about 30%, about 32%, about 34%,about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about48%, about 50% or higher from compound
 1. 57. The compound of claim 55or 56, wherein the purity of the compound is 95%, 96%, 97%, 98%, 99%,99.2%, 99.4%, 99.6%, 99.8% or higher.
 58. A method of improving cellularhealth in a subject, comprising administering to the subject a compoundof any one of claims 55-57.
 59. A method of improving sleep quality,stimulating or increasing REM sleep, or treating or preventing insomnia,desynchronosis, or a circadian rhythm sleep disorder in a subject,comprising administering to the subject a compound of any one of claims55-57.
 60. A method of treating or preventing a motor neuron disease orALS, or slowing or reversing the progression of motor neurondegeneration in a subject, comprising administering to the subject acompound of any one of claims 55-57.
 61. A method of improvingfertility, treating or preventing infertility, inducing ovulation,increasing sperm count, or increasing lactation, comprisingadministering to the subject a compound of any one of claims 55-57. 62.A method of treating or preventing kidney damage, acute kidney injury,or kidney disease, or increasing blood flow to the kidneys, comprisingadministering to the subject a compound of any one of claims 55-57. 63.A method of treating or preventing liver damage or fatty liver, ordecreasing the serum level of alanine transaminase (ALT) or aspartatetransaminase (AST) in a subject, comprising administering to the subjecta compound of any one of claims 55-57.